The p21-activated protein kinases (Paks) are likely to be key effectors for Cdc42 and Rac1; two Rho-family GTPases that regulate a variety of fundamental biological processes, including cell proliferation, shape control, migration, and stress response. Abnormalities in these processes underlie many important human diseases, including most malignancies. Because the nature of the relationship between growth factors, adaptor proteins, Rho-family GTPases, and Paks is not well defined, we propose to study in detail the mechanism(s) by which these kinases are regulated in cells by external factors and during the cell cycle. In the first two aims, we will study the regulation of Pak's biochemical activity by growth factors, adhesion, and by inflammatory cytokines. We have created two unique reagents: Pak1 mutants that selectively couple to a single GTPase, and phosphorylation-state specific antibodies that recognize only activated Pak1, which will allow us to determine precisely when and where Pak1 is activated in cells. We plan to use these reagents to assess the relative contributions of the GTPases Cdc42 and Rac, the guanine nucleotide exchange factor PIX, and the adaptor protein Nck to Pak1 activation and relocalization induced by external stimuli. In the final aim, we will examine the regulation of Pak by phosphorylation during the cell cycle. Evidence from lower eukaryotes strongly supports a regulatory role for Pak during cell cycle progression, and our preliminary data suggests this is true in mammalian cells as well. Using a combination of mapping and site-directed mutagenesis, we will determine the role of Pak1 phosphorylation during the cell cycle in fibroblasts. Achieving the aims set forth in this proposal will shed light on the activation mechanism(s) for a protein kinase which regulates at least two fundamental biologic properties: cell morphology and transcriptional responses. Understanding the molecular basis for this regulation is not only of intrinsic scientific interest but is also likely to be relevant to important human diseases such as metastatic cancer.